An antisense compound binds to or hybridizes with a nucleotide sequence in a nucleic acid (RNA or DNA) to inhibit the function (or synthesis) of the nucleic acid. Because they can hybridize with both RNA and DNA, antisense compounds can interfere with gene expression at the level of transcription, RNA processing or translation. The resulting interference leads to an inhibition of the synthesis of the protein encoded by the nucleic acid, such as the proteins of the tissues, various cellular growth factors, growth factor receptors, and oncogenes.
As discussed, e.g., in Klausner, A., Biotechnology, 8:303-304 (1990), the development of practical applications of antisense technology is hampered by a number of technical problems. Thus, natural, phosphate-linked antisense oligomer compounds are susceptible to rapid degradation by nucleases that exist in target cells and elsewhere in the body; such as exonucleases, which act on either the 3' or the 5' terminus of the nucleic acid, and endonucleases, which cleave the nucleic acid at internal phosphodiester linkages between individual nucleosides. As a result of such nuclease action, the effective half life of many administered antisense compounds is very short, necessitating the use of large, frequently administered, doses.
The high cost of producing antisense DNA or RNA on currently available DNA synthesizers is another problem. Armstrong, L., Business Week, Mar. 5, 1990, page 89, estimated the cost of producing one gram of antisense DNA to be about $100,000.
There is also a problem regarding delivery of antisense agents to targets within the body (and cell). Thus, antisense agents targeted to messenger RNA must permeate the plasma membrane and antisense agents targeted to genomic DNA must permeate both the plasma membrane and the nuclear membrane to gain access to the nucleus. The consequent need for increased hydrophobicity to enhance membrane permeability must be balanced against the need for increased hydrophilicity (water solubility) in body fluids such as the plasma and cell cytosol.
Also, oligonucleotide compounds such as antisense DNA are susceptible to steric reconfiguration around chiral phosphorous centers. This results in stability problems, too, whether the compounds are free within the body or hybridized to target nucleic acids.
To overcome the stability and drug delivery limitations, various oligonucleotide analogs have been investigated. In order to be of practical utility, such analogs should have good cell penetration properties, be resistant to nuclease degradation, have good sequence specific hybridization to target nucleic acids, and be synthesized by chemical methods that are not too difficult or costly.
Recent efforts to overcome the foregoing problems and prepare antisense compounds that are stable, nuclease resistant, relatively inexpensive to manufacture and which can be delivered to and hybridized with nucleic acid targets throughout the body have involved synthesizing oligonucleotide analogs that consist of oligonucleoside sequences with internucleoside linkages that differ from the `normal` internucleoside phosphodiester linkage, either by introducing modifications in the phosphodiester structure or by using non-phosphate internucleoside linkages that approximate the length and orientation of the normal phosphodiester internucleoside linkage. Uhlman, E. and Peyman, A., Chemical Reviews, 9(4):544-584 (1990).
Among the modified phosphodiester linkages that have been reported are phosphorothioates, alkylphosphotriesters, methylphosphonates and alkylphosphoramidates. Also, a variety of non-ionic oligonucleoside sequences containing non-phosphate internucleoside linkages, such as carbonate, acetate, carbamate, sulfone, sulfoxide, sulfonamide and dialkyl- or diaryl- silyl derivatives have been synthesized and reported. More recently, chimeric oligonucleotide analogs comprising nucleoside linkages containing two carbon atoms and one nitrogen atom or one oxygen atom, as well as those containing three carbon atoms, have been reported. See, e.g., International Patent Publication WO 9202534.